Combination therapy for reducing side effects using cannabinoid receptor ligands

ABSTRACT

The present application describes a method of treating conditions treatable with a CB 2  cannabinoid receptor agonist with reduced CB 1 -mediated side effects using a combination therapy comprising a CB 2  agonist and a CB 1  ligand, which can be a CB 1  antagonist or a CB 1  inverse agonist.

Present invention seeks priority from U.S. Provisional PatentApplication 60/980,967, filed on Oct. 18, 2007, which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD AND BACKGROUND

The present application relates to a method of treating pain states withreduced side effects resulting from a therapeutic use of a CB₂cannabinoid receptor agonist comprising a combination therapy consistingof a CB₂ cannabinoid receptor agonist in an amount effective to obtain atherapeutic effect, and a CB₁ cannabinoid receptor ligand to the subjectin an amount effective to block the adverse effects but not toantagonize the therapeutic effect of the cannabinoid receptor agonist.

(−)-Δ⁹-Tetrahydrocannabinol (Δ⁹-THC), the major psychoactive constituentof marijuana, exerts a broad range of biological effects through itsinteractions with two cannabinoid (CB) receptor subtypes, CB₁ and CB₂.CB₁ receptors are highly expressed in the central nervous system and toa lesser degree in the periphery in a variety of tissues of thecardiovascular and gastrointestinal systems. By contrast, CB₂ receptorsare most abundantly expressed in multiple lymphoid organs and cells ofthe immune system, including spleen, thymus, tonsils, bone marrow,pancreas and mast cells.

The psychotropic effects caused by Δ⁹-THC and other nonselective CBagonists are mediated by CB₁ receptors. Activation of the CB₁ receptorby administration of a CB₁ agonist produces a number of undesirablephysiological and behavioral effects. In the central nervous system,activation of the CB₁ receptor can result in a variety of psychotropiceffects such as euphoria, sedation, catalepsy, paranoia, panic andanxiety. CB₁ activation also negatively impacts cognitive function,leading to a loss of short-term memory, poor executive function andimpaired learning. CB₁ activation also produces physiological effectsthat manifest themselves outside the central nervous system such ashypothermia, increased heart rate, decreased blood pressure, and drymouth. The undesirable effects mediated by CB1 activation may negativelyimpact the usefulness to a patient of any medication that displays someability to activate the CB1 receptor. The undesirable effects resultingfrom activation of the CB₁ receptor with a CB₁ agonist may be blocked,inhibited, or prevented by administration of a selective CB₁ antagonistor inverse agonist.

Several lines of evidence support the assertion that CB₂ receptors playa role in analgesia. For example, Zimmer et al. have reported that thenonselective cannabinoid agonist Δ⁹-THC retains some analgesic efficacyin CB₁ receptor knockout mice (Zimmer, A., et al., Proc. Nat. Acad.Sci., 1999, 96, 5780-5785). HU-308 is one of the first highly selectiveCB₂ agonists identified that elicits an antinociceptive response in therat formalin model of persistent pain (Hanus, L., et al., Proc. Nat.Acad. Sci., 1999, 96, 14228-14233). The CB₂-selective cannabiniod ligandAM-1241 exhibits robust analgesic efficacy in animal models of acutethermal pain (Malan, T. P., et al., Pain, 2001, 93, 239-245; Ibrahim, M.M., et al., Proc. Nat. Acad. Sci., 2005, 102(8), 3093-3098), persistentpain (Hohmann, A. G., et al., J. Pharmacol. Exp. Ther., 2004, 308,446-453), inflammatory pain (Nackley, A. G., et al., Neuroscience, 2003,119, 747-757; Quartilho, A. et al., Anesthesiology, 2003, 99, 955-60),and neuropathic pain (Ibrahim, M. M., et al., Proc. Nat. Acad. Sci.,2003, 100, 10529-10533). The CB₂-selective partial agonist GW405833,also known as L768242, is efficacious in rodent models of neuropathic,incisional, and both chronic and acute inflammatory pain (Valenzano, K.J., et al., Neuropharmacology, 2005, 48, 658-672 and Clayton, N., etal., Pain, 2002, 96, 253-260). The analgesic effects induced by theseCB₂-selective ligands are blocked by CB₂ and not by CB₁ receptorantagonists.

CB₂ receptors are present in tissues and cell types associated withimmune functions and CB₂ receptor mRNA is expressed by human B cells,natural killer cells, monocytes, neutrophils, and T cells (Galiegue etal., Eur. J. Biochem., 1995, 232, 54-61). Studies with CB₂ knockout micehave suggested a role for CB₂ receptors in modulating the immune system(Buckley, N. E., et al., Eur. J. Pharmacol. 2000, 396, 141-149).Although immune cell development and differentiation are similar inknockout and wild type animals, the immunosuppressive effects of Δ⁹-THCare absent in the CB₂ receptor knockout mice, providing evidence for theinvolvement of CB₂ receptors in immunomodulation. As such, selective CB₂agonists may be useful for the treatment of autoimmune diseasesincluding but not limited to multiple sclerosis, rheumatoid arthritis,systemic lupus, myasthenia gravis, type I diabetes, irritable bowelsyndrome, psoriasis, psoriatic arthritis, and hepatitis; and immunerelated disorders including but not limited to tissue rejection in organtransplants, gluten-sensitive enteropathy (Celiac disease), asthma,chronic obstructive pulmonary disease, emphysema, bronchitis, acuterespiratory distress syndrome, allergies, allergic rhinitis, dermatitis,and Sjogren's syndrome.

Microglial cells are considered to be the immune cells of the centralnervous system (CNS) where they regulate the initiation and progressionof immune responses. They are quiescent and resting having a ramifiedmorphology as long as the CNS is healthy. Microglia express a variety ofreceptors enabling them to survey the CNS and respond to pathologicalevents. Insult or injury to the CNS leads to microglial cell activation,which is characterized by various morphological changes allowingresponse to the lesion. Ramifications are retracted and microglia aretransformed into amoeboid-like cells with phagocytic function. They canproliferate, rapidly migrate to the site of injury, and produce andrelease cytokines, chemokines and complement components (Watkins L. R.,et al., Trends in Neuroscience, 2001, 24(8), 450; Kreutzberg, G. W.,Trends Neurosci., 1996, 19, 312-318). CB₂ receptor expression onmicroglia is dependent upon inflammatory state with higher levels of CB₂found in primed, proliferating, and migrating microglia relative toresting or fully activated microglial (Carlisle, S. J., et al. Int.Immunopharmacol., 2002, 2, 69). Neuroinflammation induces many changesin microglia cell morphology and there is an upregulation of CB₂receptors and other components of the endocannabinoid system. It isconceivable that CB₂ receptors may be more susceptible topharmacological effects during neuroinflammation (Walter, L., Stella,N., Br. J. Pharmacol. 2004, 141, 775-785). Neuroinflammation occurs inseveral neurodegenerative diseases, and induction of microglial CB₂receptors has been observed (Carrier, E. J., et al., Current DrugTargets—CNS & Neurological Disorders, 2005, 4, 657-665). Thus, CB₂ligands may be clinically useful for the treatment of neuroinflammation.

CB₂ receptor expression has been detected in perivascular microglialcells within normal, healthy human cerebellum (Nunez, E., et al.,Synapse, 2004, 58, 208-213). Perivascular cells are immunoregulatorycells located adjacent to CNS blood vessels and, along with parenchymalmicroglia and astrocytes, they play a pivotal role in maintaining CNShomeostasis and blood-brain barrier functionality (Williams, K., et al.,Glia, 2001, 36, 156-164). CB₂ receptor expression has also been detectedon cerebromicrovascular endothelial cells, which represent a maincomponent of the blood-brain barrier (Golech, S. A., et al., Mol. Brain.Res., 2004, 132, 87-92). A recent report demonstrated that CB₂ receptorexpression is up-regulated in the brains of macaques with simianimmunodeficiency virus-induced encephalitis (Benito, C., et al., J.Neurosci. 2005, 25(10), 2530-2536). Thus, compounds that affect CB₂signaling may protect the blood-brain barrier and be clinically usefulin the treatment of neuroinflammation and a variety of neuroinflammatorydisorders including retroviral encephalitis, which occurs with humanimmunodeficiency virus (HIV) infection in the CNS.

Multiple sclerosis is common immune-mediated disease of the CNS in whichthe ability of neurons to conduct impulses becomes impaired throughdemyelination and axonal damage. The demyelination occurs as aconsequence of chronic inflammation and ultimately leads to a broadrange of clinical symptoms that fluctuate unpredictably and generallyworsen with age. These include painful muscle spasms, tremor, ataxia,motor weakness, sphincter dysfunction, and difficulty speaking (Pertwee,R. G., Pharmacol. Ther. 2002, 95, 165-174). The CB₂ receptor isup-regulated on activated microglial cells during experimentalautoimmune encephalomyelitis (EAE) (Maresz, K., et al., J. Neurochem.2005, 95, 437-445). CB₂ receptor activation prevents the recruitment ofinflammatory cells such as leukocytes into the CNS (Ni, X., et al.,Multiple Sclerosis, 2004, 10, 158-164) and plays a protective role inexperimental, progressive demyelination (Arevalo-Martin, A.; et al., J.Neurosci., 2003, 23(7), 2511-2516), which are critical features in thedevelopment of multiple sclerosis. Thus, CB₂ receptor agonists mayprovide a unique treatment for demyelinating pathologies.

Alzheimer's disease is a chronic neurodegenerative disorder accountingfor the most common form of elderly dementia. Recent studies haverevealed that CB₂ receptor expression is upregulated in neuriticplaque-associated microglia from brains of Alzheimer's disease patients(Benito, C., et al., J. Neurosci., 2003, 23(35), 11136-11141). In vitro,treatment with the CB₂ agonist JWH-133 abrogated β-amyloid-inducedmicroglial activation and neurotoxicity, effects that can be blocked bythe CB₂ antagonist SR144528 (Ramirez, B. G., et al., J. Neurosci. 2005,25(8), 1904-1913). CB₂ agonists possessing both anti-inflammatory andneuroprotective actions thus may have clinical utility in treatingneuroinflammation and in providing neuroprotection associated with thedevelopment of Alzheimer's disease.

Increased levels of epithelial CB₂ receptor expression are observed inhuman inflammatory bowel disease tissue (Wright, K., et al.,Gastroenterology, 2005, 129, 437-453). Activation of CB₂ receptorsre-established normal gastrointestinal transit after endotoxicinflammation was induced in rats (Mathison, R., et al., Br. J.Pharmacol. 2004, 142, 1247-1254). CB₂ receptor activation in a humancolonic epithelial cell line inhibited TNF-α-induced interleukin-8(IL-8) release (Ihenetu, K. et al., Eur. J. Pharmacol. 2003, 458,207-215). Chemokines released from the epithelium, such as theneutrophil chemoattractant IL-8, are upregulated in inflammatory boweldisease (Warhurst, A. C., et al., Gut, 1998, 42, 208-213). Thus,administration of CB₂ receptor agonists represents a novel approach forthe treatment of inflammation and disorders of the gastrointestinaltract including but not limited to inflammatory bowel disease, irritablebowel syndrome, secretory diarrhea, ulcerative colitis, Crohn's diseaseand gastroesophageal reflux disease (GERD).

Hepatic fibrosis occurs as a response to chronic liver injury andultimately leads to cirrhosis, which is a major worldwide health issuedue to the severe accompanying complications of portal hypertension,liver failure, and hepatocellular carcinoma (Lotersztajn, S., et al.,Annu. Rev. Pharmacol. Toxicol., 2005, 45, 605-628). Although CB₂receptors were not detectable in normal human liver, CB₂ receptors wereexpressed liver biopsy specimens from patients with cirrhosis.Activation of CB₂ receptors in cultured hepatic myofibroblasts producedpotent antifibrogenic effects (Julien, B., et al., Gastroenterology,2005, 128, 742-755). In addition, CB₂ knockout mice developed enhancedliver fibrosis after chronic administration of carbon tetrachloriderelative to wild-type mice. Administration of CB₂ receptor agonistsrepresents a unique approach for the treatment of liver fibrosis.

CB₂ receptors are involved in the neuroprotective and anti-inflammatorymechanisms induced by the interleukin-1 receptor antagonist (IL-1ra)(Molina-Holgado, F., et al., J. Neurosci., 2003, 23(16), 6470-6474).IL-1ra is an important anti-inflammatory cytokine that protects againstischemic, excitotoxic, and traumatic brain insults. CB₂ receptors play arole in mediating these neuroprotective effects indicating that CB₂agonists may be useful in the treatment of traumatic brain injury,stroke, and in mitigating brain damage.

Cough is a dominant and persistent symptom of many inflammatory lungdiseases, including asthma, chronic obstructive pulmonary disease, viralinfections, and pulmonary fibrosis (Patel, H. J., et al., Brit. J.Pharmacol., 2003, 140, 261-268). Recent studies have provided evidencefor the existence of neuronal CB₂ receptors in the airways, and havedemonstrated a role for CB₂ receptor activation in cough suppression(Patel, H. J., et al., Brit. J. Pharmacol., 2003, 140, 261-268 andYoshihara, S., et al., Am. J. Respir. Crit. Care Med., 2004, 170,941-946). Both exogenous and endogenous cannabinoid ligands inhibit theactivation of C-fibers via CB₂ receptors and reduce neurogenicinflammatory reactions in airway tissues (Yoshihara, S., et al., J.Pharmacol. Sci. 2005, 98(1), 77-82; Yoshihara, S., et al., Allergy andImmunology, 2005, 138, 80-87). Thus, CB₂-selective agonists may haveutility as antitussive agents for the treatment of pulmonaryinflammation, chronic cough, and a variety of airway inflammatorydiseases including but not limited to asthma, chronic obstructivepulmonary disease, and pulmonary fibrosis.

Artherosclerosis is a chronic inflammatory disease and is a leadingcause of heart disease and stroke. CB₂ receptors have been detected inboth human and mouse atherosclerotic plaques. Administration of lowdoses of THC in apolipoprotein E knockout mice slowed the progression ofatherosclerotic lesions, and these effects were inhibited by theCB₂-selective antagonist SR144528 (Steffens, S., et al., Nature, 2005,434, 782-786). Thus, compounds with activity at the CB₂ receptor may beclinically useful for the treatment of atherosclerosis.

CB₂ receptors are expressed on malignant cells of the immune system andtargeting CB₂ receptors to induce apoptosis may constitute a novelapproach to treating malignancies of the immune system. Selective CB₂agonists induce regression of malignant gliomas (Sanchez, C., et al.,Cancer Res., 2001, 61, 5784-5789), skin carcinomas (Casanova, M. L., etal., J. Clin. Invest., 2003, 111, 43-50), and lymphomas (McKallip, R.J., et al., Blood, 2002, 15(2), 637-634). Thus, CB₂ agonists may haveutility as anticancer agents against tumors of immune origin.

In view of this evidence, compounds that are selective agonists for theCB2 receptor over the CB₁ receptor are potential therapeutic agents forthe treatment of a variety of disorders including inflammatory pain,inflammatory disorders, immune disorders, neurological disorders,neurodegeneration, cancer, respiratory disorders, cardiovasculardisorders, osteoporosis, obesity, and diabetes.

Pain alleviation occurs at doses of a CB₂ selective agonist that do notcause or may to a very limited extent cause undesirable effectsassociated with CB₁ receptor activation. At higher doses, however, drugconcentrations of a CB₂ selective agonist may reach sufficiently highlevels so as to begin to activate CB₁ receptors, due to some residualweak CB₁ activity of the CB₂ selective agent producing ataxia andcatalepsy (Valenzano et al., Neuropharmacology, Vol. 48 pages 658-672,2005). Therefore, at higher doses CB₂ selective agonists may begin todisplay the aforementioned undesirable effects due to residual CB₁activation.

In view of the aforementioned evidence, a combination therapyco-administering a CB₂ selective agonist and a CB₁ selective antagonistor inverse agonist can provide the therapeutic benefit of alleviatingpain through activation of CB₂ receptors while concomitantly preventingthe undesirable adverse effects due to the residual weak activation ofthe CB₁ receptor. This combination therapy provides a safer, moretolerable and more effective method of treating pain, or any otherdisorder mediated through CB₂ receptors, with reduced incidence of abuseliability.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the effect of(Z)-N-(5-tert-butyl-3-butylthiazol-2(3H)-ylidene)-5-chloro-2-methoxybenzamide,a CB₂ selective agonist, in skin incision pain model.

FIG. 1B shows the effect of(Z)-N-(3-(2-methoxyethyl)-4,5-dimethylthiazol-2(3H)-ylidene)-2,2,3,3-tetramethylcyclopropanecarboxamide,a CB₂ selective agonist, in skin incision pain model.

FIG. 2 depicts the effect of co-dosing of(Z)-N-(5-tert-butyl-3-butylthiazol-2(3H)-ylidene)-5-chloro-2-methoxybenzamide(30 μmol/kg, intraperitoneally), a CB₂ agonist and rimonabant (30μmol/kg, intraperitoneally), on skin incision pain model.

FIG. 3 depicts the effects of co-dosing of(Z)-N-(3-(2-methoxyethyl)-4,5-dimethylthiazol-2(3H)-ylidene)-2,2,3,3-tetramethylcyclopropanecarboxamide(μmol/kg, intraperitoneally), a CB₂ selective agonist, and rimonabant(30 μmol/kg, intraperitoneally), on motor activity.

FIG. 4 represents the effects of co-dosing(Z)-N-(5-tert-butyl-3-butylthiazol-2(3H)-ylidene)-5-chloro-2-methoxybenzamide,a CB₂ selective agonist (30 μmol/kg, intraperitoneally), and rimonabant(30 μmol/kg, intraperitoneally) on motor activity.

FIG. 5 summarizes the effects of co-dosing of(Z)-N-(3-(2-methoxyethyl)-4,5-dimethylthiazol-2(3H)-ylidene)-2,2,3,3-tetramethylcyclopropanecarboxamide(30 μmol/kg, intraperitoneally), a CB₂ selective agonist, and rimonabant(30 μmol/kg, intraperitoneally), on grip force.

FIG. 6 shows the effects on mean arterial pressure after co-dosing of(Z)-N-(3-(2-methoxyethyl)-4,5-dimethylthiazol-2(3H)-ylidene)-2,2,3,3-tetramethylcyclopropanecarboxamide(1 mg/kg, intravenously), a CB₂ selective agonist, and rimonabant (3.2mg/kg, intravenously).

FIG. 7 shows the comparison of efficacy and side effects of(Z)-N-(5-tert-butyl-3-butylthiazol-2(3H)-ylidene)-5-chloro-2-methoxybenzamide,a CB₂ selective agonist, alone and in combination with rimonabant. Skinincision refers to the postoperative skin incision pain model. LMArefers to locomotor activity model.

DETAILED DESCRIPTION OF THE INVENTION

The present application claims a method for treating a subject sufferingfrom a condition treatable with a CB₂ selective receptor agonist using acombination therapy comprising administering a CB₂ receptor agonist inan amount effective to obtain a therapeutic effect, and a CB₁ receptorligand to the subject in an amount effective to block any CB₁ mediatedadverse effects but not to antagonize the therapeutic effect of the CB₂selective receptor agonist. It is intended that the selective CB₁cannabinoid receptor ligand may be a CB₁ receptor antagonist, or a CB₁receptor inverse agonist. It is also intended that the combinationtherapy comprising the CB₂ cannabinoid receptor agonist and the CB₁receptor ligand, may be represented by the separate administration of apharmaceutical composition containing the selective CB₂ cannabinoidreceptor agonist, and a pharmaceutical composition containing the CB₁receptor ligand, each pharmaceutical composition further comprising atherapeutically acceptable carrier. On the other hand, the combinationtherapy comprising the CB₂ cannabinoid receptor agonist and the CB₁receptor ligand, may be represented by a single pharmaceuticalcomposition comprising both compounds and therapeutically acceptablecarrier, in which the CB₁ receptor ligand may be a CB₁ receptorantagonist, or a CB₁ receptor inverse agonist.

The present invention also relates to a method of reducing the side andunwanted effects of a CB₂ receptor agonist in a subject suffering from acondition treatable with a CB₂ receptor agonist, comprisingadministering a selective CB₂ receptor agonist and administering a CB₁receptor ligand to the subject in an amount effective to block the sideand unwanted effects but not to antagonize the therapeutic effect of theCB₂ receptor agonist. It is intended that the selective CB₁ receptorligand may be a CB₁ receptor antagonist, or a CB₁ receptor inverseagonist.

Conditions treatable with the combination therapy of the invention,comprising administration of a CB2 selective agonist and a CB1 selectiveligand (antagonist or inverse agonist), include pain, specificallyneuropathic pain, nociceptive pain, or inflammatory pain. Othertreatable conditions of the present invention include but are notlimited to inflammatory disorders, immune disorders, neurologicaldisorders, cancer of the immune system, respiratory disorders, obesity,diabetes, and cardiovascular disorders. Evidence to support said claimis present herein.

Several lines of evidence support the assertion that CB₂ receptors areassociated with a variety of cells and tissues, and having a role inmany physiological mechanisms that make selective agonists of the CB₂receptor important therapeutic agents for a large variety of disorders.

The analgesic effects induced by selective CB₂ agonists are blocked byCB₂ and not by CB₁ receptor antagonists. CB₂ agonists are useful in thetreatment of pain states comprising neuropathic pain, inflammatory painor nociceptive pain, arising from disorders such as cancer, HIV,multiple sclerosis, diabetic neuropathy, post-herpetic neuralgia,arthritis, osteoarthritis, rheumatoid arthritis, surgical procedures andothers. Some residual CB₁ receptor activation may induce unwanted sideeffects such as ataxia, catalepsy, and euphoria. Administration orcombination of a selective CB₂ receptor agonist with a selective CB₁receptor antagonist/inverse agonist provides a way to induce analgesiawithout the liability of the undesired side effects, which includetolerance, dependence, addiction, sedation, euphoria, dysphoria, memoryimpairment, hallucination, depression, dry mouth, increased heart rate,dizziness and headache among others.

CB₁ selective antagonist and inverse agonist molecules are well-known tothose skilled in the art. Examples of representative CB₁ selectiveantagonists and inverse agonists can be found in the followingliterature references: Muccioli, Expert Opin. Ther. Pat. (2006) 16, pp1405-1423; Barth, Ann. Rep. Med. Chem. (2005), 40, pp 103-118; Hertzog,Expert Opin. Ther. Pat. (2004) 14, pp 1435-1452. The examples containedwithin the foregoing citations are meant to be illustrative of CB₁selective antagonists and inverse agonists and do not limit the scope ofCB₁ selective antagonists and inverse agonists contemplated as part ofthe invention.

Rimonabant, also known as SR141716A and5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide,is a particular CB₁ antagonist/inverse agonist contemplated as part ofthe invention.

Taranabant, also known as MK-0364 andN-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-{[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamideis a particular CB₁ antagonist/inverse agonist contemplated as part ofthe invention.

CB₂ agonist molecules are well-known to those skilled in the art.Examples of representative CB₂ agonists can be found in the followingliterature reference: Cheng, Expert Opin. Invest. Drugs Vol. 16, pages951-965 (2007). For the purpose of the present invention, examples ofsuch CB₂ selective agonists that are known in the scientific literatureinclude but are not limited to AM1241, HU308, JWH-133, GW405833,(Z)-N-(5-tert-butyl-3-butylthiazol-2(3H)-ylidene)-5-chloro-2-methoxybenzamide,and(Z)-N-(3-(2-methoxyethyl)-4,5-dimethylthiazol-2(3H)-ylidene)-2,2,3,3-tetramethylcyclopropanecarboxamide.The foregoing examples are meant to be illustrative of CB₂ selectiveagonists and do not limit the scope of CB₂ selective agonistscontemplated as part of the invention.

A CB₂ agonist is herein defined as a molecule that binds to the CB2receptor and causes activation of the receptor as measured in one ormore assays designed to detect said receptor activation. Such assays canbe either in vitro assays or in vivo assays. Typical in vitro assays formeasuring CB₂ receptor activation include, but are not limited to, themeasurement of cyclic AMP accumulation (Mukherjee, Eur. J. Pharmacol.Vol. 505, pages 1-9, 2004), the measurement of Ca²⁺ flux using afluorometric imaging plate reader (Mukherjee, Eur. J. Pharmacol. Vol.505, pages 1-9, 2004), the measurement of GTPγS binding (MacLennan etal., British J. Pharm. Vol. 124 pages 619-622, 1998)) and themeasurement of ERK or MAP kinase (Yao, Br. J. Pharmacol. Vol. 149, pages145-154, 2006). In vivo assays for detecting CB₂ receptor activationinclude the measurement of activity in a pain model upon administrationof a test compound, said activity being reversed by pretreatment with aCB₂ selective antagonist or inverse agonist. This protocol is well-knownto those skilled in the art and can be found in the following list ofreferences: Clayton, Pain Vol. 96, pages 253-260, 2002; Ibrahim, Proc.Natl. Acad. Sci. Vol. 100, pages 10529-10533, 2003; LaBuda, Eur. J.Pharmacol. Vol. 527, pages 172-174, 2005. Pain models used to detect CB₂receptor activation may be any of those described herein or known tothose skilled in the art. As defined herein a CB₂ agonist need not showCB₂ receptor activation in both an in vitro and in vivo assay but mustshow activity in at least one in vitro or in vivo assay designed tomeasure such activity. Under certain artificial assay conditions a CB₂receptor agonist such as AM1241 may appear to not activate the CB₂receptor in vitro (Yao, Br. J. Pharmacol. Vol. 149, pages 145-154, 2006)yet it may still activate the CB₂ receptor in vivo as measured using oneof the in vivo assays described above.

A CB₂ agonist may possess varying degrees of selectivity relative toactivity at the CB₁ receptor as measured in biological assays. A CB₂selective agonist is herein defined as a ligand that binds to oractivates the CB₂ receptor with at least about 100 times or greaterpotency than it binds to or activates the CB₁ receptor. It is notnecessary that a molecule be considered selective in both binding andfunctional (activation) assays to be a CB₂ selective agonist. Bindingpotency is routinely reported as the Ki, with a lower Ki value equatingwith greater potency. Thus, a CB₂ selective agonist possesses a CB₂binding Ki that is at least about 100 times or more lower than its CB₁binding Ki. Potency to activate a receptor is also routinely reported asthe EC₅₀, with the lower EC₅₀ value equating with a greater potency. TheEC₅₀ value is also associated with a measured maximum response(efficacy) in an assay relative to a reference standard. CP55,940 is acommonly used reference standard agonist at the CB₂ and CB₁ receptors,and its maximum response is set at 100%. Test compounds thus maydemonstrate full, partial or no substantial efficacy relative toCP55,940. Full efficacy represents a response of greater than or equalto about 70%. Partial efficacy represents a response of approximately20-70%. Substantially no efficacy represents a response of less thanabout 20%. Since the EC₅₀ value is the approximate concentration thatgives a 50% response relative to the maximum for the particular testagent, a test agent with a lower intrinsic efficacy may yield a lowerEC₅₀ value when compared with an agent that possesses higher intrinsicefficacy. When dealing with test agents that possess substantiallydifferent intrinsic efficacies at CB₂ and CB₁ receptors, establishingthe degree of CB₂ selectivity may not be possible simply by comparingEC₅₀ values, since these each may be based upon substantially differentmaxima. Thus, a meaningful mathematical calculation of CB₂ selectivitycannot be made by comparing the CB₂ and CB1 EC₅₀s for a representativetest agent that potently activates the CB₂ receptor with full efficacyand weakly activates the CB₁ receptor with no substantial efficacy. Yetsuch a compound would generally be appreciated by one skilled in the artas a CB₂ selective agonist. A CB₂ selective agonist, thus, produces aparticular degree of activation of the CB₂ receptor at a concentrationat least about 100 times or more lower than the concentration to elicitsubstantially the same degree of activation of the CB₁ receptor relativeto a reference standard agonist like CP55,940. In other words, theconcentration to activate the CB2 receptor is at least about 100 timesor more lower than the equi-effective concentration to activate the CB₁receptor. The equi-effective concentration refers to the concentrationthat produces substantially the same degree of activation of the CB₁receptor as the CB₂ receptor, wherein this degree of activation couldrange from 20% to 100%. Also, a compound that partially or fullyactivates the CB₂ receptor and does not substantially activate the CB₁receptor is considered a CB₂ selective agonist.

For purposes of the present invention a CB₂ selective agonist possessesa CB₂ binding Ki of less than or equal to 100 nM, preferably less thanor equal to 10 nM, and a selectivity ratio of 100 or greater relative tothe CB₁ receptor.

The CB₂ selective agonist of the present invention also possesses a CB₂binding Ki of less than or equal to 10 nM and a selectivity ratio of1000 or greater relative to the CB receptor.

For purposes of the invention a CB₂ selective agonist possesses a CB₂binding Ki of less than or equal to 1 nM and a selectivity ratio of 100or greater, preferably 1000, more preferably 10,000, relative to the CB₁receptor.

To determine the selectivity (Ki) of the compounds of the presentapplication for CB₂ receptors relative to CB₁ receptors, radioligandbinding assays are performed, which are described herein.

For CB₂ radioligand binding assays, HEK293 cells stably expressing humanCB₂ receptors were grown until a confluent monolayer was formed.Briefly, the cells were harvested and homogenized in TE buffer (50 mMTris-HCl, 1 mM MgCl₂, and 1 mM EDTA) using a polytron for 2×10 secondbursts in the presence of protease inhibitors, followed bycentrifugation at 45,000×g for 20 minutes. The final membrane pellet wasre-homogenized in storage buffer (50 mM Tris-HCl, 1 mM MgCl₂, and 1 mMEDTA and 10% sucrose) and frozen at −78° C. until used. Saturationbinding reactions were initiated by the addition of membrane preparation(protein concentration of 5 μg/well for human CB₂) into wells of a deepwell plate containing [³H]CP-55,940 (120 Ci/mmol, a nonselective CBagonist commercially available from Tocris) in assay buffer (50 mM Tris,2.5 mM EDTA, 5 mM MgCl₂, and 0.5 mg/mL fatty acid free BSA, pH 7.4).After 90 min incubation at 30° C., binding reaction was terminated bythe addition of 300 μl/well of cold assay buffer followed by rapidvacuum filtration through a UniFilter-96 GF/C filter plates (pre-soakedin 1 mg/mL BSA for 2 hours). The bound activity was counted in aTopCount using Microscint-20. Saturation experiments were conducted withtwelve concentrations of [³H]CP-55,940 ranging from 0.01 to 8 nM.Competition experiments were conducted with 0.5 nM [³H]CP-55,940 andfive concentrations of displacing ligands in concentrations selectedfrom the range of 0.01 nM to 10 μM. The addition of 10 μM unlabeledCP-55,940 (Tocris, Ellisville, Mo.) was used to assess nonspecificbinding.

For CB₁ radioligand binding assay HEK293 human CB₁ membranes werepurchased from Perkin Elmer. Binding was initiated by the addition ofmembranes (8-12 μg per well) into wells (Scienceware 96-well DeepWellplate, VWR, West Chester, Pa.) containing [³H]CP-55,940 (120 Ci/mmol,Perkin Elmer, Boston, Mass.) and a sufficient volume of assay buffer (50mM Tris, 2.5 mM EDTA, 5 mM MgCl₂, and 0.5 mg/mL fatty acid free BSA, pH7.4) to bring the total volume to 250 μL. After incubation (30° C. for90 minutes), binding was terminated by the addition of 300 μL per wellof cold assay buffer and rapid vacuum filtration (FilterMate CellHarvester, Perkin Elmer, Boston, Mass.) through a UniFilter-96 GF/Cfilter plate (Perkin Elmer, Boston, Mass.) (pre-soaked in 0.3% PEI atleast 3 hours), followed by five washes with cold assay buffer. Thebound activity was counted in the TopCount using Microscint-20 (bothfrom Perkin Elmer, Boston, Mass.). Competition experiments wereconducted with 1 nM [³H]CP-55,940 and five concentrations (1 nM to 10μM) of displacing ligands. The addition of 10 μM unlabeled CP-55,940(Tocris, Ellisville, Mo.) was used to assess nonspecific binding.

The compounds used in the examples of the present application preferablybind to CB₂ receptors, therefore are selective ligands for the CB₂receptor, as indicated by the relative values of Ki for each of thereceptors:(Z)-N-(5-tert-butyl-3-butylthiazol-2(3H)-ylidene)-5-chloro-2-methoxybenzamide(compound A), a CB₂ selective agonist, has a Ki for CB₂ receptors of 1.8nM, and a Ki for CB₁ receptors of 3670 nM;(Z)-N-(3-(2-methoxyethyl)-4,5-dimethylthiazol-2(3H)-ylidene)-2,2,3,3-tetramethylcyclopropanecarboxamide(compound B), the other CB₂ selective agonist used in the presentapplication, has a Ki for CB₂ receptors of 0.6 nM, and a Ki for CB₁receptors or 273 nM.

In terms of effectiveness, it is intended that the CB₂ selective agonistof the present invention partially or fully activate the CB2 receptor asdefined herein.

In terms of effectiveness, it is intended that the CB₂ selective agonistof the present invention possesses a CB₂ EC₅₀ of less than or equal to100 nM, preferably 10 nM, and fully activates the CB₁ receptor with anEC₅₀ of 10,000 nM or greater. Additionally, the CB₂ selective agonist ofthe invention possesses a CB₂ EC₅₀ of less than or equal to 10 nM andfully activates the CB₁ receptor with an EC₅₀ of 1,000 nM or greater.

It is intended that the CB₂ selective agonist of the invention possessesa CB₂ EC₅₀ of less than or equal to 1 nM and fully activates the CB₁receptor with an EC₅₀ of 100 nM or greater, preferably 1000 nM, morepreferably 10,000 nM.

The CB₂ selective agonist of the invention also possesses a CB₂ EC₅₀ ofless than or equal to 100 nM and partially activates the CB₁ receptor,wherein the equi-effective concentration to activate the CB₁ receptor isat least 100 times higher than that to activate the CB₂ receptor. Alsoincluded in the present invention are CB₂ selective agonists thatpossess a CB₂ EC₅₀ of less than or equal to 10 nM and that partiallyactivate the CB₁ receptor, wherein the equi-effective concentration toactivate the CB₁ receptor is at least 100 to 1000 times higher than thatto activate the CB₂ receptor.

In one embodiment of the invention a CB₂ selective agonist possesses aCB₂ EC₅₀ of less than or equal to 1 nM and partially activates the CB₁receptor, wherein the equi-effective concentration to activate the CB₁receptor is at least 100 times, preferably 1,000, more preferably 10,000higher than that to activate the CB₂ receptor. For purposes of theinvention a CB₂ selective agonist possesses a CB₂ EC₅₀ of less than orequal to 100 nM, preferably less than or equal to 10 nM, more preferablyless than or equal to 1 nM, and does not substantially activate the CB₁receptor in vitro.

For purposes of the present invention a CB₂ selective agonist produces aresponse in an in vivo pain model of 30% or more. Said response may beinhibited by a CB₂ antagonist or by a CB₂ inverse agonist, but not by aCB₁ antagonist or CB₁ inverse agonist.

A CB₁ selective antagonist or CB₁ selective inverse agonist is definedherein as a molecule that inhibits the activation of the CB₁ receptor oralternatively reduces the basal level of activity of the CB₁ receptor.Assays to detect CB₁ antagonist and inverse agonist activity arewell-known to those skilled in the art, a list of such assays beingfound in the following references: Pertwee, Life Sci. Vol. 76, pages1307-1324, 2005; Muccioli, Curr. Med. Chem. Vol. 12, pages 1361-1394,2005.

A CB₂ antagonist or CB₂ inverse agonist is defined herein as a moleculethat inhibits the activation of the CB2 receptor or alternativelyreduces the basal level of activity of the CB₂ receptor. Assays todetect CB₂ antagonist and inverse agonist activity are well-known tothose skilled in the art, such assays being found or referred to in thefollowing references: Rinaldi-Carmona, J. Pharmacol. Exp. Ther. Vol.284, pages 644-650, 1998; Muccioli, Exp. Opin. Ther. Pat. Vol. 16, pages1405-1423, 2006; Raitio, Curr. Med. Chem. Vol. 12 pages 1217-1237, 2005.

Typical CB₂ antagonists/inverse agonists that can be utilized todemonstrate CB₂ receptor activation in vivo include SR144528 also knownas{N-[(1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide}and AM630 also known as(6-iodo-2-methyl-1-(2-morpholinoethyl)-1H-indol-3-yl)(4-methoxyphenyl)methanone.

Several methods are available to test the efficacy of compounds inalleviating pain; among them are the Incision Model of PostoperativePain, Spinal Nerve Ligation Model of Neuropathic Pain, andCapsaicin-induced secondary mechanical hypersensitivity.

Adult male Sprague-Dawley rats (250-300 g body weight, Charles RiverLaboratories, Portage, Mich.) were used. Animal handling andexperimental protocols were approved by the Institutional Animal Careand Use Committee (IACUC) at Abbott Laboratories. For all surgicalprocedures, animals were maintained under halothane anesthesia (4% toinduce, 2% to maintain), and the incision sites were sterilized using a10% povidone-iodine solution prior to and after surgeries.

A skin incision model of postoperative pain was produced using theprocedures described in Brennan et al., 1996, Pain, 64, 493. All ratswere anesthetized with isoflurane delivered via a nose cone. Right hindpaw incision was performed following sterilization procedures. Theplantar aspect of the left hind paw was placed through a hole in asterile plastic drape. A 1-cm longitudinal incision was made through theskin and fascia of the plantar aspect of the hind paw, starting 0.5 cmfrom the proximal edge of the heel and extending towards the toes, theplantar muscle was elevated and incised longitudinally leaving themuscle origin and insertion points intact. The skin was then closed withtwo mattress sutures (5-0 nylon). After surgery, animals were thenallowed to recover for 2 hours, at which time tactile allodynia wasassessed as described below. To evaluate the anti-nociceptive effects,animals were i.p. administered vehicle or test compound 90 minutesfollowing skin incision and tactile allodynia was typically assessed 30minutes after compound administration.

Tactile allodynia was measured using calibrated von Frey filaments(Stoelting, Wood Dale, Ill.) as described in Chaplan, S. R., F. W. Bach,J. W. Porgrel, J. M. Chung and T. L. Yaksh, 1994, Quantitativeassessment of tactile allodynia in the rat paw, J. Neurosci. Methods,53, 55. Rats were placed into inverted individual plastic cage(20×12.5×20 cm) on top of a suspended wire mesh grid, and acclimated tothe test chambers for 20 minutes. The von Frey filaments were appliedperpendicularly from underneath the cage through openings in the wiremesh floor directly to an area within 1-3 mm (immediately adjacent) ofthe incision, and then held in this position for approximately 8 secondswith enough force to cause a slight bend in the filament. Positiveresponses included an abrupt withdrawal of the hind paw from thestimulus, or flinching behavior immediately following removal of thestimulus. A 50% withdrawal threshold was determined using an up-downprocedure as described in Dixon, W. J., 1980, Efficient analysis ofexperimental observations, Ann. Rev. Pharmacol. Toxicol., 20, 441. Theeffect on paw withdrawal latency of test compounds compared to vehiclecontrol represents the ability of the test compound to reduce allodynia.An increase in the paw withdrawal latency represents an anti-allodyniceffect (i.e., a decrease in pain).

A model of spinal nerve ligation-induced (SNL model) neuropathic painwas originally described by Kim and Chung (Kim, S. H. and J. M. Chung,Pain Vol. 50, page 355, 1992) and can also be used to test the compoundsof the present application The left L5 and L6 spinal nerves of the ratare isolated adjacent to the vertebral column and tightly ligated with a5-0 silk suture distal to the DRG, and care is taken to avoid injury ofthe L4 spinal nerve. Sham animals undergo the same procedure, butwithout nerve ligation. All animals are allowed to recover for at leastone week and not more than three weeks prior to assessment of tactileallodynia. Only animals with a baseline threshold score of less that4.25 g are used in this study, and animals demonstrating motor deficitare excluded. Tactile allodynia thresholds are also assessed in severalcontrol groups, including naive, sham-operated, and saline infusedanimals as well as in the contralateral paws of nerve-injured animals.

In the capsaicin-induced secondary mechanical hypersensitivity assay,animals receive capsaicin at 10 μg in 10 μl of vehicle (10% ethanol and2-hydroxypropyl cyclodextrin) by intraplantar injection into the centerof the right hind paw. Secondary mechanical hyperalgesia is measured atthe heel away from the site of injection at 180 min following capsaicin(Joshi et al 2006, Neuroscience 143, 587-596). Compounds are injected(i.p.) 30 min before testing (150 min post-capsaicin). Tactile allodyniais measured as described above.

Adverse effects can be assessed by measurements of Grip Force (GF)Behavior. Measurements of hind limb grip force are conducted byrecording the maximum compressive force exerted on the hind limb straingauge setup, in a commercially available grip force measurement system(Columbus Instruments, Columbus, Ohio). During testing, animals aregently restrained by grasping around its rib cage and then allowed tograsp the wire mesh frame (10×12 cm²) attached to the strain gauge. Theexperimenter then moves the animal in a rostral-to-caudal directionuntil the grip is broken. Each animal is sequentially tested twice atapproximately 2-3 min interval to obtain a raw mean grip force(CF_(max)). This raw mean grip force data is in turn converted to amaximum hindlimb compressive force (CF_(max)) (gram force)/kg bodyweight for each animal. A group mean±S.E.M. for CF_(max)/kg body weightis calculated. A group of age-matched naïve animals are added to eachexperiment and the data obtained from the different dose groups for eachcompound—or compound combination—being tested are compared to the naïvegroup (assigned as being 100% normal). A reduction in hindlimb gripforce for a test compound compared to the effect of vehicle is a measureof adverse effects; the greater the reduction in grip force, the greaterthe adverse effect. All experiments evaluating drug effects in thismodel are conducted in a randomized blinded fashion. The statisticalanalysis of variance (ANOVA) is carried out using GraphPad Prism(GraphPad Software, Inc., San Diego, Calif.). Bonferroni's multiplecomparison test is performed as a post hoc comparison.

Adverse effects may also be determined by the Measurement of LocomotorActivity. Spontaneous activity is assessed in an open-field environment(42 (length)×42 (base)×40 cm (height); Piper Plastics, Libertyville,Ill.) situated inside Versamax/Digiscan monitors, each equipped withinfrared sensors (AccuScan Instruments, Inc., Columbus, Ohio) in a dimlyilluminated test room. Following administration of test compounds orvehicle control, rats are individually placed into the test chambers andhorizontal (locomotion) activity is recorded for 30 min. A reduction inhorizontal motor activity for a test compound compared to the effect ofvehicle is a measure of adverse effects; the greater the reduction inactivity, the greater the adverse effect. Data is analyzed by ANOVAfollowed by Fisher's Protected Least significant difference (PLSD)analysis as a post hoc comparison (JMP statistic database; SASInstitute, Inc., Cary, N.C.). A p<0.05 was considered significant.

The principal embodiment of the present application is a method fortreating a subject suffering from a condition treatable with a CB₂selective agonist comprising administering a CB₂ selective agonist in anamount sufficient to obtain a therapeutic effect in combination with aCB₁ antagonist in an amount sufficient to block any residual adverseeffect from the CB₂ selective agonist but not reduce the therapeuticeffect of said CB₂ selective agonist. This combination therapy can beeffective by separately administering each of the two compounds at thesame time or one immediately after the other in doses sufficient toobtain the desired therapeutic effect. This combination therapy can alsobe effective by combining both compounds in the same pharmaceuticalcomposition in amounts effective to obtain the desired therapeuticeffect.

In another embodiment, the present application provides a pharmaceuticalcomposition that comprises the compounds of the present invention. Thepharmaceutical compositions comprise compounds of the present inventionformulated separately or together with one or more non-toxicpharmaceutically acceptable carriers. The pharmaceutical compositions ofthis invention can be administered to humans and other mammals orally,rectally, parenterally, intracisternally, intravaginally, topically (asby powders, ointments or drops), bucally or as an oral or nasal spray.The term “parenterally,” as used herein, refers to modes ofadministration that include intravenous, intramuscular, intraperitoneal,intrasternal, subcutaneous and intraarticular injection and infusion.

The term “pharmaceutically acceptable carrier,” as used herein, means anon-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type. Someexamples of materials which can serve as pharmaceutically acceptablecarriers are sugars such as, but not limited to, lactose, glucose andsucrose; starches such as, but not limited to, corn starch and potatostarch; cellulose and its derivatives such as, but not limited to,sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients such as, but notlimited to, cocoa butter and suppository waxes; oils such as, but notlimited to, peanut oil, cottonseed oil, safflower oil, sesame oil, oliveoil, corn oil and soybean oil; glycols; such as propylene glycol; esterssuch as, but not limited to, ethyl oleate and ethyl laurate; agar;buffering agents such as, but not limited to, magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol, and phosphate buffer solutions, aswell as other non-toxic compatible lubricants such as, but not limitedto, sodium lauryl sulfate and magnesium stearate, as well as coloringagents, releasing agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants can also be present inthe composition, according to the judgment of the formulator.

Pharmaceutical compositions of this invention for parenteral injectioncomprise pharmaceutically acceptable sterile aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions as well as sterilepowders for reconstitution into sterile injectable solutions ordispersions just prior to use. Examples of suitable aqueous andnonaqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols (such as glycerol, propylene glycol, polyethyleneglycol and the like), vegetable oils (such as olive oil), injectableorganic esters (such as ethyl oleate) and suitable mixtures thereof.Proper fluidity can be maintained, for example, by the use of coatingmaterials such as lecithin, by the maintenance of the required particlesize in the case of dispersions and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms can be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid and the like. It may also be desirableto include isotonic agents such as sugars, sodium chloride and the like.Prolonged absorption of the injectable pharmaceutical form can bebrought about by the inclusion of agents, which delay absorption such asaluminum monostearate and gelatin.

In some cases, in order to prolong the effect of the drug(s), it isdesirable to slow the absorption of the drug(s) from subcutaneous orintramuscular injection. This can be accomplished by the use of a liquidsuspension of crystalline or amorphous material with poor watersolubility, or by dissolving or suspending the drug(s) in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending upon the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions, which are compatiblewith body tissues.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms, the activecompound(s) may be mixed with at least one inert, pharmaceuticallyacceptable carrier or excipient, such as sodium citrate or dicalciumphosphate and/or a) fillers or extenders such as starches, lactose,sucrose, glucose, mannitol and silicic acid; b) binders such ascarboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,sucrose and acacia; c) humectants such as glycerol; d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates and sodium carbonate; e) solutionretarding agents such as paraffin; f) absorption accelerators such asquaternary ammonium compounds; g) wetting agents such as cetyl alcoholand glycerol monostearate; h) absorbents such as kaolin and bentoniteclay and i) lubricants such as talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate and mixturesthereof. In the case of capsules, tablets and pills, the dosage form mayalso comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such carriers as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike.

The solid dosage forms of tablets, dragees, capsules, pills and granulescan be prepared with coatings and shells such as enteric coatings andother coatings well-known in the pharmaceutical formulating art. Theymay optionally contain opacifying agents and may also be of acomposition such that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes.

The active compound(s) can also be in micro-encapsulated form, ifappropriate, with one or more of the above-mentioned carriers.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirs. Inaddition to the active compounds, the liquid dosage forms may containinert diluents commonly used in the art such as, for example, water orother solvents, solubilizing agents and emulsifiers such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethyl formamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan andmixtures thereof. Besides inert diluents, the oral compositions may alsoinclude adjuvants such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring and perfuming agents.

Dosage forms for topical administration of a compound of this inventioninclude powders, sprays, ointments and inhalants. The active compoundmay be mixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives, buffers or propellants, which maybe required. Opthalmic formulations, eye ointments, powders andsolutions are also contemplated as being within the scope of thisinvention.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this application will depend upon the activity of theparticular compound, the route of administration, the severity of thecondition being treated and the condition and prior medical history ofthe patient being treated.

When used in the above or other treatments, a therapeutically effectiveamount of one of the compounds of the present invention can be employedin pure form or, where such forms exist, in pharmaceutically acceptablesalt, ester or prodrug form. The phrase “therapeutically effectiveamount” of the compound of the invention means a sufficient amount ofthe compound to treat disorders, at a reasonable benefit/risk ratioapplicable to any medical treatment. It will be understood, however,that the total daily usage of the compounds and compositions of thepresent invention will be decided by the attending physician within thescope of sound medical judgment. The specific therapeutically effectivedose level for any particular patient will depend upon a variety offactors including the disorder being treated and the severity of thedisorder; activity of the specific compound employed; the specificcomposition employed; the age, body weight, general health, sex and dietof the patient; the time of administration, route of administration, andrate of excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts.

The following examples are only intended to illustrate and not to limitthe scope of the present application.

EXAMPLE 1 CB₂ Selective Agonists Effective in Skin Incision Model

Using the skin incisional model of postoperative pain, the effects ofcompound A, CB₂ agonist(Z)-N-(5-tert-butyl-3-butylthiazol-2(3H)-ylidene)-5-chloro-2-methoxybenzamide,on allodynia was assessed two hours after surgery. FIG. 1A shows thatthe withdrawal threshold for the incisional paw recovered upon i.p.administration of 10 and 30 μmol/kg of CB₂ agonist A. FIG. 1B showssimilar results with compound B, CB₂ agonist(Z)-N-(3-(2-methoxyethyl)-4,5-dimethylthiazol-2(3H)-ylidene)-2,2,3,3-tetramethylcyclopropanecarboxamide,re-establishing withdrawal threshold at 10 and 30 μmol/kg i.p. Theseresults indicate the potent effects obtained with CB₂ agonists in thisrat pain model.

EXAMPLE 2 Effects of CB₂ Agonist and CB₁ Antagonist on Analgesia in SkinIncision Model

Using the skin incisional model of postoperative pain, the effects ofCB₂ agonist A were evaluated after the administration of vehicle or theCB₁ antagonist rimonabant. This effect was compared with theadministration of vehicle after the administration of vehicle or the CB1antagonist rimonabant. A first dose, consisting of CB₁ antagonistrimonabant (30 μmol/kg i.p.) or a vehicle (5% DMSO/PEG), wasadministered 15 minutes before a second dose, consisting ofadministration of CB₂ agonist A (30 μmol/kg i.p.) or vehicle. Allodyniatesting was conducted 30 minutes after administration of the seconddose, either CB2 agonist A or vehicle. FIG. 2 shows that either the twoadministrations of vehicle or the administration of vehicle 15 minutesafter the CB₁ antagonist rimonabant did not ameliorate the tactileallodynia in the operated rats. FIG. 2 also shows that CB₂ agonist Aadministered after vehicle, or the CB1 antagonist rimonabant, was ableto induce analgesia in this model. These results indicate that the levelof analgesia induced by the CB₂ agonist was not affected by the prioradministration of a CB₁ antagonist.

EXAMPLE 3 Effects of CB₁ Antagonist and CB₂ Agonist B on SpontaneousLocomotor Activity

FIG. 3 shows that CB₂ agonist B significantly reduces spontaneouslocomotor activity when administered to rats at 30 and 45 μmol/kg i.p.(solid bars). This reduction in locomotor activity was eliminated when30 μmol/kg i.p CB₁ antagonist rimonabant was administered 15 minutesbefore the administration of CB₂ agonist B. All tests were conducted 30minutes after the administration of CB2 agonist B. These resultsindicate that a higher dose of the CB₂ agonist B did not reduce therats' spontaneous activity when administered with a CB₁ antagonist.These effects also indicate that the reduction in locomotor activity byCB2 agonist B results from its residual activation of the CB1 receptor.Accordingly, co-dosing CB₂ agonist B with the CB₁ antagonist rimonabantsignificantly reduced the adverse effects.

EXAMPLE 4 Effects of CB₁ Antagonist and CB₂ Agonist A on SpontaneousLocomotor Activity

Using the locomotor activity assay, the effects of CB₂ agonist A wereevaluated after the administration of vehicle or the CB₁ antagonistrimonabant. This effect was compared with the administration of vehicleafter the administration of vehicle or the CB1 antagonist rimonabant. Afirst dose, consisting of CB₁ antagonist rimonabant (30 μmol/kg i.p.) ora vehicle (5% DMSO/PEG), was administered 15 minutes before a seconddose, consisting of administration of CB₂ agonist A (30 μmol/kg i.p.) orvehicle. Locomotor testing was conducted 30 minutes after administrationof the second dose, either CB2 agonist A or vehicle. FIG. 4 shows thatCB₁ antagonist rimonabant did not affect horizontal locomotor activityof vehicle. CB₂ agonist A administered after vehicle decreasedhorizontal locomotor activity by 91%. CB₁ antagonist rimonabant was ableto block said decrease when administered 15 minutes prior to CB₂ agonistA. These effects indicate that the reduction in locomotor activity byCB2 agonist A results from its residual activation of the CB1 receptor.FIG. 4 shows that administration of CB₁ antagonist and CB₂ agonisteliminates unwanted side effects induced by residual CB₁ activity of theCB₂ agonist.

EXAMPLE 5 Effect of CB₁ Antagonist and CB₂ Agonist on Grip ForceBehavior

Using the grip force behavior assay, the effects of CB₂ agonist B wereevaluated after the administration of vehicle or the CB₁ antagonistrimonabant. This effect was compared with the administration of vehicleafter the administration of vehicle or the CB1 antagonist rimonabant. Afirst dose, consisting of CB₁ antagonist rimonabant (30 μmol/kg i.p.) ora vehicle (5% DMSO/PEG), was administered 15 minutes before a seconddose, consisting of administration of CB₂ agonist B (30 μmol/kg i.p.) orvehicle. Grip force behavior testing was conducted 30 minutes afteradministration of the second dose, either CB2 agonist B or vehicle. FIG.5 shows CB₂ agonist B induced reduction of grip force by 42% (30 μmol/kgi.p). This effect was completely blocked by prior administration of CB₁antagonist rimonabant. As shown in FIG. 5, rimonabant had no effect onthe grip force when administered prior to vehicle. These effectsindicate that the reduction in grip force by CB2 agonist B results fromits residual activation of the CB1 receptor. FIG. 5 shows thatadministration of CB₁ antagonist and CB₂ agonist eliminates unwantedside effects induced by residual CB₁ activity of the CB₂ agonist.

EXAMPLE 6 Effect of CB₁ Antagonist and CB₂ Agonist Co-Administration onMean Arterial Pressure (MAP) and Heart Rate (HR)

Male Sprague-Dawley rats were anesthetized with the long actingbarbiturate, Inactin. Catheters were placed in the femoral artery formeasurement of MAP and HR. Additional catheters were placed in thefemoral vein for compound administration and saline infusion to maintainhydration. Following a 30-minute control period, vehicle (PEG400),compound B (vehicle: PEG400; 2 ml/kg) or CB₂ agonist B (vehicle: PEG400;2 ml/kg) plus CB₁ antagonist rimonabant (vehicle: PEG400; 1 ml/kg) wasadministered intravenously over a 30-minute infusion. When administeredsimultaneously, compound B and rimonabant were infused individuallyusing separate syringes. The doses tested here were 1.0 mg/kg forcompound B and 3.2 mg/kg for rimonabant. Data is expressed as mean from3 rats±sem. FIG. 6 demonstrates the effects on mean arterial pressure(MAP) of administration of vehicle, compound B alone, and compound Bwith rimonabant. These results demonstrate that the combinationtreatment of compound B with rimonabant is devoid of an effect on bloodpressure relative to vehicle whereas the treatment with compound B hasan effect to reduce blood pressure. These effects indicate that thereduction in blood pressure by CB2 agonist B results from its residualactivation of the CB1 receptor. FIG. 6 shows that administration of CB₁antagonist and CB₂ agonist eliminates unwanted side effects induced byresidual CB₁ activity of the CB₂ agonist.

EXAMPLE 7 A Comparison of the Efficacy and Side Effects for a CB₂Agonist Alone and a CB₂ AGONIST dosed with a CB₁ Antagonist

FIG. 7 shows the effect in the skin incision pain model and thelocomotor side effect assay for CB₂ agonist compound A, and compound Aplus CB₁ antagonist rimonabant with the data expressed as the maximumpossible effect obtainable in these assays. Compound A and compound Aco-dosed with rimonabant show equivalent efficacy in the skin incisionpain model. Compound A plus rimonabant shows much reduced side effectsin the locomotor assay compared with compound A dosed alone. Compound Awas administered at a dose of 30 μmol/kg i.p and rimonabant wasadministered at a dose of 30 μmol/kg i.p. 15 minutes before compound A.

1. A method for treating a subject suffering from a condition treatablewith a selective CB₂ cannabinoid receptor agonist using a combinationtherapy comprising administering a selective CB₂ receptor agonist in anamount effective to obtain a therapeutic effect, and a CB₁ cannabinoidreceptor ligand in an amount effective to block any adverse effectsmediated by the CB₁ receptor, but not to antagonize the therapeuticeffect of the CB₂ receptor agonist.
 2. The method of claim 1, whereinthe selective CB₁ cannabinoid receptor ligand is a CB₁ receptorantagonist.
 3. The method of claim 1, wherein the selective CB₁cannabinoid receptor ligand is a CB₁ receptor inverse agonist.
 4. Themethod of claim 1 wherein the condition is selected from the groupcomprising neuropathic pain, nociceptive pain, and inflammatory pain. 5.The method of claim 1 wherein the condition is selected from the groupcomprising inflammatory disorders, immune disorders, neurologicaldisorders, cancers of the immune system, respiratory disorders, obesity,diabetes, and cardiovascular disorders.
 6. The combination therapy ofclaim 1, wherein the CB₂ receptor agonist and the CB1 receptor ligandare administered as separate pharmaceutical compositions including apharmaceutically acceptable carrier.
 7. The combination therapy of claim1, wherein the CB₂ receptor agonist and the CB₁ receptor ligand areadministered together as one pharmaceutical composition including apharmaceutically acceptable carrier.
 8. A method of reducing theCB₁-mediated side and unwanted effects of a CB₂ cannabinoid receptoragonist in a subject suffering from a condition treatable with aselective CB₂ agonist comprising administering a selective CB₂ agonistand administering a CB₁ ligand to the subject in an amount effective toblock the side and unwanted effects but not to antagonize thetherapeutic effect of the CB₂ agonist.
 9. The method of claim 8, whereinthe CB₁ cannabinoid receptor ligand is a CB₁ receptor antagonist. 10.The method of claim 8, wherein the CB₁ cannabinoid receptor ligand is aCB₁ receptor inverse agonist.
 11. The method of claim 8 wherein thecondition is selected from the group comprising neuropathic pain,nociceptive pain, and inflammatory pain.
 12. The method of claim 8wherein the condition is selected from the group comprising inflammatorydisorders, immune disorders, neurological disorders, cancers of theimmune system, respiratory disorders, obesity, diabetes, andcardiovascular disorders.
 13. A composition comprising a selective CB₂receptor agonist in an amount effective to produce a therapeutic effect,a CB₁ receptor ligand in an amount effective to antagonize side andadverse effects produced by the CB₂ agonist, but not to antagonize thetherapeutic effect of the CB₂ agonist, and a pharmaceutically acceptablecarrier.
 14. The composition of claim 13, wherein the CB₁ receptorligand is a CB₁ receptor antagonist.
 15. The composition of claim 13,wherein the CB₁ receptor ligand is a CB₁ receptor inverse agonist.